Project Summary/Abstract Autism spectrum disorder (ASD) is a significant cause of morbidity and loss of developmental potential in children. An increasing number of genetic and environmental factors are being identified that contribute to ASD. Two major cellular pathways that have been implicated in both genetic and environmental causes of ASD involve mTOR signaling and GABAergic neurotransmission. In this project, we propose to investigate a mechanistic link between these pathways that may explain both the age dependence and male sex predominance of core ASD symptoms and comorbidities. In this project, we propose to test the hypothesis that autism-associated mTOR pathway activation causes differential dysregulation of GABAergic neurotransmission in the immature versus the mature brain. Further, we hypothesize that this dysregulation will be more pronounced in males than in females. We will examine our hypotheses through pursuit of the following Specific Aims. Specific Aim 1: To test the hypothesis that mTOR pathway activation accelerates the development of hyperpolarizing GABAA receptor function in the immature brain. Cultured cortical neurons and transgenic mice with mTOR pathway activation, either via heterozygous loss of the Pten or Tsc2 genes or via expression of a constitutively active form of mTOR, will be used. Specific Aim 2: To test the hypothesis that mTOR pathway activation interferes with maintenance of hyperpolarizing GABAA receptor function in the mature brain. In Specific Aim 2, the studies of Aim 1 will be repeated in mature neuronal cultures and in adult mice. Specific Aim 3: To define the role of the transcriptional repressor REST (RE1-Silencing Transcription factor) in the age-dependent dysregulation of GABAergic neurotransmission by mTOR pathway activation. We will utilize cortical neuronal cultures and lentivirus-mediated manipulation of REST function in vivo to investigate the involvement of REST in the bidirectional regulation of KCC2 expression by mTOR pathway activation. As a result of these studies, we will be able to identify the differential effects of mTOR pathway activation on inhibitory neurotransmission in immature versus mature and in male versus female brain, thereby suggesting pathways for development of novel, targeted interventions for ASD and its comorbidities.